1. Field of the Invention
The present invention generally relates to a thermal cycler, such as for a nucleic acid amplification apparatus. Specifically, the present invention may relate to a nucleic acid amplification apparatus that thermally cycles a fluid containing nucleic acid, by causing the fluid to flow in a channel running through temperature zones set at different temperatures, thereby amplifying the nucleic acid.
2. Description of the Related Art
To efficiently duplicate and amplify a very small amount of template DNA, a polymerase chain reaction (PCR) method is commonly used. The PCR method achieves amplification of DNA of interest through repetition of a thermal cycle including the following steps (1) to (3). The step (1) is a denaturing step for thermally denaturing double-stranded DNA into single-stranded DNA, which functions as a template. The step (2) is an annealing step for annealing the template and primers that are complementary to the template. The step (3) is an extension step for synthesizing double-stranded DNA, by forming a DNA strand that is complementary to the template from the primers with a thermally stable DNA polymerase.
The steps are generally performed by controlling the temperatures and reaction times to which a reaction fluid is subjected, whereby an amplification reaction occurs in the reaction fluid. Typically, double-stranded DNA is thermally denatured into single-stranded DNA, which functions as a template, at a temperature of about 94° C. Primers are annealed to single-stranded DNA at a temperature of about 65° C. A DNA strand that is complementary to the template is synthesized with a DNA polymerase at a temperature of about 72° C.
An apparatus exists that automatically performs the PCR method by changing the temperature of a reaction fluid in an Eppendorf tube with a heater and a cooler. The reaction fluid contains template DNA, primers, deoxyribonucleoside triphosphate (dNTP), a DNA polymerase, and the like. The apparatus has wells formed in an aluminum block, and may control the temperature of the block, thereby controlling the temperature of the Eppendorf tubes inserted into the wells.
In the PCR method, thermal cycling may need to be performed under accurate control of temperature. However, when the PCR method is performed as the batch reaction described above, thermal fluctuation of the reaction system may considerably increase as the scale of the system increases. For this reason, the degree to which the scale of the system can be increased is generally restricted.
Japanese Patent Laid-Open Nos. 06-30776 and 07-075544 and Kopp M U; Mello A J; Manz A., Science, 1998, 280, 5366, pp 1046-1048 disclose a continuous flow PCR method with which it is claimed that thermal cycling can be performed under accurate control of temperature, and increasing the scale of the system can also be achieved. In the method, a reaction fluid containing a DNA polymerase, template DNA, primer DNA, dNTP, and the like, is thermally cycled by flowing the fluid through a channel running through a heated zone and a cooled zone, thereby performing the PCR.
FIG. 2 of PCT Japanese Translation Patent Publication No. 2001-521622 shows a PCR method in which a current is passed through a fluid flowing through a channel, thereby generating joule heat. The fluid has a temperature that depends on dissipation of heat from the channel. The fluid at a position in the channel also has a temperature that depends on the cross-sectional area of the channel at the position. The time for which the fluid flows at a certain temperature depends on the length of the channel. Thus, since the fluid at a position in the channel has a temperature that depends on the geometry of the channel and heat dissipation from the channel to the environment, the method may not provide sufficiently accurate temperature control.
The PCR method described in Kopp M U; Mello A J; Manz A., Science, 1998, 280, 5366, pp 1046-1048 is conducted with the following configuration. Three temperature zones are arranged in a plane in the order of 94° C., 73° C., and 55° C. zones. A portion of a channel in the 73° C. zone functions as a first intermediate portion in which a fluid rapidly flows from the 94° C. zone to the 55° C. zone. In contrast, another portion of the channel in the 73° C. zone functions as a second intermediate portion in which the fluid flows from the 55° C. zone to the 94° C. zone at a rate slower than that in the first intermediate portion, to provide sufficient time for extending DNA. To address these competing requirements, which include providing as short a passing time as possible as well as sufficient time for DNA extension in the respective intermediate portions at 73° C., the portions are provided with different channel lengths, whereby the fluid takes different amounts of time to pass through the portions. This configuration increases the length of the channel, thereby increasing flow resistance of the channel. To flow a PCR fluid through a long channel, pressure may be applied to the fluid. However, application of an excessively high negative pressure to a fluid can cause the fluid to boil, because the boiling point of the fluid is decreased under the pressure. Application of a high positive pressure to a fluid may require taking measures for preventing the fluid from leaking, which can increase the size of cartridges and the costs of producing such cartridges. A longer channel may also adsorb a larger portion of template DNA molecules, decreasing amplification yield. A longer channel may also require a larger plane area where the channel is to be arranged, and hence size reduction of an apparatus employing the PCR method may not be achieved.